Methods and compositions for treatment of alzheimer&#39;s disease and other neurodegenerative disorders

ABSTRACT

A method for treating Alzheimer&#39;s disease (AD) by administering to an AD patient a PKC activator, such as a bryostatin-1, without administering a NMDA receptor antagonist, such as memantine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/914,399 filed June 28, 2020, which is a continuation ofPCT/US2019/032762, filed May 17, 2019 and claims priority from U.S.Provisional Application No. 62/673,590, filed May 18, 2018, which isherein incorporated by reference in its entirety.

BACKGROUND

All references cited herein are expressly incorporated by reference.

Treatments of Alzheimer's disease (AD) have been based on immunotherapyfor pathologic hallmarks of the autopsy AD brain, amyloid plaques andneurofibrillary tangles generated by hyperphosphorylated tau protein.Other therapeutic strategies have focused on enhancement or blockade ofneurotransmitters at synaptic junctions (Reisberg et al. N Engl J Med.2003 Apr. 3;348(14):1333-41). While the latter have generated drugs withsome symptomatic efficacy, the approved drugs have thus far not beeneffective in preventing, improving, and/or reversing the progressiveneurodegeneration that ultimately causes major cognitive dysfunction andfunctional decline in AD patients (Farlow et al., Clin. Ther. 2010July;32(7):1234-51. doi: 10.1016; Farlow et al., BMC Neurology 2011 vol.II, article 57; and Farlow, Neurology 2005 65:S1-S4). This lack ofefficacy is likely related to the lack of close correlation demonstratedby plaques and tangles with the degree of cognitive deficits.

On the other hand, cognitive deficits have been shown to correlateclosely with the loss of synapses as measured directly or indirectly atautopsy (Terry et al., Ann Neurol. 1991 October;30(4):572-80). Aneffective therapeutic for AD should be directed toward, not only thecauses, but also the consequences of the characteristic progressiveneurodegeneration. Based on the synaptic loss in AD, there would be asignificant benefit in a therapeutic strategy that can restore lostsynapses in AD brains.

SUMMARY

Methods of treating Alzheimer's disease (AD) are described hereinincluding administering to an AD patient in need thereof a proteinkinase C (PKC) activator, wherein the patient is not administered anNMDA receptor antagonist. In embodiments, the PKC activator isbryostatin or analog thereof. In embodiments, the PKC activator isbryostatin-1.

Methods of treating Alzheimer's disease are also described hereinincluding administering to an AD patient in need thereof a PKCactivator, wherein the treatment excludes administration of a NMDAreceptor antagonist. In embodiments, the PKC activator is bryostatin oranalog thereof. In embodiments, the PKC activator is bryostatin-1.

Methods of treating Alzheimer's disease are also described hereinincluding administering to an AD patient in need thereof bryostatin-1wherein the patient is not also administered memantine.

Methods of treating Alzheimer's disease are also described hereinincluding administering to an AD patient in need thereof no more than orless than about 30, 35, or 40 mcg (or range therein) of bryostatin-1wherein the patient is not also administered memantine. In embodiments,the patient is administered less than 40 mcg bryostatin-1. In someembodiments the patient is administered about 20 mcg bryostatin-1.

Methods of treating Alzheimer's disease are also described hereinincluding administering to an AD patient in need thereof about 20 mcg or25 mcg (or range therein) of bryostatin-1 wherein the treatment excludesmemantine.

Methods of treating Alzheimer's disease are also described hereinincluding administering to an AD patient in need thereof no more than orless than about 30, 35, or 40 mcg (or range therein) of bryostatin-1wherein the patient is not receiving memantine treatment. In someembodiments the patient is administered 15-40 mcg bryostatin-1. In otherembodiments, the patient is administered less than 40 mcg bryostatin-1

Methods of treating Alzheimer's disease are also described hereinincluding administering to an AD patient in need thereof about 20 mcg or25 mcg (or range therein) of bryostatin-1 wherein the patient is notreceiving memantine treatment. In some embodiments the patient isadministered about 20 mcg bryostatin-1.

Methods of treating Alzheimer's disease are also described hereinincluding administering to an AD patient in need thereof about 15 mcg toabout 25 mcg (or range therein) of bryostatin-1 wherein the treatmentexcludes memantine and provides cognitive improvement. In someembodiments the patient is administered about 20 mcg bryostatin-1.

Methods of treating Alzheimer's disease are also described hereinincluding administering to a patient in need thereof about 20 mcg or 25mcg (or range therein) of bryostatin-1 wherein the treatment excludesmemantine and is provided for at least or more than 4, 5, or 6 weeks.

Each of the methods described herein may further include theadministration of a compound selected from the group consisting ofgalantamine, donepezil, rivastigimine, physostigmine, tacrine, huperzineA, ladostigil, and combinations thereof.

In some embodiments, the patient in need of treatment is a patientdiagnosed with mild Alzheimer's disease. In other embodiments, thepatient is diagnosed with mild to moderate Alzheimer's disease.

In some embodiments, the patient in need of treatment is a patientdiagnosed with moderate Alzheimer's disease. In other embodiments, thepatient is diagnosed with moderate to severe Alzheimer's disease. Inother embodiments, the patient in need of treatment is a patientdiagnosed with severe Alzheimer's disease.

In some embodiments, the patient is diagnosed with early onsetAlzheimer's disease. In other embodiments, the patient is diagnosed withlate onset Alzheimer's disease.

Compositions for treating Alzheimer's Disease are also described hereinand include at least one PKC activator, the composition being free ofany NMDA receptor antagonist. In some embodiments, the PKC activator isbryostatin or an analogue thereof. In particular embodiments, the PKCactivator is bryostatin-1. In embodiments, the compositions are free ofmemantine.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent and Trademark Officeupon request and payment of the necessary fee.

Some specific embodiments of the disclosure may be understood byreferring, in part, to the following description and the accompanyingdrawings.

FIG. 1 includes graphs showing the SIB change from baseline compared toplacebo for the modified intent-to-treat (mITT) population and also theCompleters population treated with Bryostatin.

FIG. 2 is a graph showing the SIB change from baseline compared toplacebo 30 days post dosing in the mITT population.

FIG. 3 is a graph showing the SIB change from baseline compared toplacebo 30 days post dosing in the Completers population.

FIG. 4 includes graphs showing the SIB change from baseline compared toplacebo for the mITT population treated with bryostatin with or withoutmemantine.

FIG. 5 includes graphs showing the SIB change from baseline compared toplacebo for the Completers population treated with bryostatin with orwithout memantine.

FIG. 6 includes graphs showing the ADCS-ADL-SIV efficacy in the mITTpopulation and the Completers population.

FIG. 7 includes graphs showing the sustained improvement of the SIBversus baseline and placebo 30 days post dosing in the Completerspopulation.

FIG. 8 is a graph showing the Body Surface Area analysis of a bryostatindose.

DETAILED DESCRIPTION

In general, the present disclosure provides methods for treatingAlzheimer's disease using PKC activators in the absence of any NMDAreceptor antagonist, such as memantine. As used herein, the term“protein kinase C activator” (i.e., “PKC activator”) refers to asubstance that increases the rate of the reaction catalyzed by proteinkinase C, upregulates the expression of PKC (e.g., upregulates theexpression of PKCα, PKC βII, PKC γ and/or PKC ε), or otherwisefacilitates the activation of PKC. In certain embodiments, the PKCactivator may be any of bryostatins 1-20, a bryolog, neristatin, apolyunsaturated fatty acid, or a combination of two or more of any ofthe foregoing substances.

As used herein, the term “NMDA receptor antagonist” refers to asubstance that antagonizes (i.e., inhibits) the action of NMDAreceptors. Some examples of NMDA receptor antagonists include memantine,drug combinations containing memantine, dextromethorphan, ketamine,phencyclidine (PCP), methoxetamine (MXE), and nitrous oxide (N₂O). Anyone or all of the foregoing NMDA receptor antagonists may be excludedfrom the method of treating AD.

In certain embodiments, the present disclosure provides methodscomprising administering to a human subject with Alzheimer's disease apharmaceutically effective amount of a PKC activator withoutadministering any NMDA receptor antagonist, such as memantine. The PKCactivator may be administered as part of a composition suitable foradministration to a human subject. The compositions used in the methodsof the present disclosure may be administered via any suitable route,such as, for example, orally, intraperitoneally, subcutaneously,intranasally, buccally, trans-dermally intramuscularly, intrarectally,intravenously, or by oral inhalation.

In some embodiments, the present disclosure provides methods comprisingadministering to a human subject with Alzheimer's disease a compositionconsisting essentially of, or containing exclusively, a pharmaceuticallyeffective amount of a PKC activator in a pharmaceutically suitablecarrier, optionally along with one or more pharmaceutically inactiveauxiliary agents. Thus, the composition preferably does not include asecond active agent, particularly a NMDA receptor antagonist.Compositions and methods consisting essentially of a PKC activatorexclude additional active compounds, such as memantine.

Bryostatins, in particular, may be used as a PKC activator in themethods of the present disclosure. The term “bryostatins” is hereinmeant to also include the numerous known analogues thereof, unlessotherwise specified. The bryostatins are a family of naturally occurringmacrocyclic compounds originally isolated from marine bryozoa.Currently, there are about 20 known natural bryostatins which sharethree six-membered rings designated A, B and C, and which differ mainlyin the nature of their substituents at C7 (OR^(A)) and C20 (R^(B)). Arepresentative generic structure of the bryostatins is provided below:

Bryostatin 1 and analogues (derivatives) of bryostatin 1 are describedin U.S. Pat. No. 4,560,774, the contents of which are hereinincorporated by reference in their entirety. Some examples ofbryostatins that may be used as a PKC activator in methods of thepresent disclosure include bryostatin 1, bryostatin 2, bryostatin 3,bryostatin 4, bryostatin 5, bryostatin 6, bryostatin 7, bryostatin 8,bryostatin 9, bryostatin 10, bryostatin 11, bryostatin 12, bryostatin13, bryostatin 14, bryostatin 15, bryostatin 16, bryostatin 17bryostatin 18, bryostatin 19, and bryostatin 20.

Analogues of bryostatins, commonly referred to as bryologs, may or maynot be used in the methods of the present disclosure. Bryologs arestructural analogues of bryostatin. While bryostatin has two pyran ringsand one 6-membered cyclic acetal, in most bryologs one of the pyrans ofbryostatin is replaced with a second 6-membered acetal ring. Thismodification reduces the stability of bryologs, relative to bryostatin,for example, in both strong acid or base, but has little significance atphysiological pH. Bryologs also have a lower molecular weight (rangingfrom about 600 to 755), as compared to bryostatin (988), a propertywhich may facilitate transport across the blood-brain barrier. Examplesof suitable bryologs include, but are not limited to analogs andderivatives of bryostatins, such as those described in U.S. Pat. Nos.6,624,189, 7,256,286 and 8,497,385, the disclosures of which areincorporated herein by reference in their entirety.

Another example of suitable PKC activators include potassium channelactivators, such as, for example, diazoxide. In certain embodiments,neristatins, such as neristatin 1, may be used in the methods of thepresent disclosure for treating Alzheimer's disease. Other suitable PKCactivators include, but are not limited to,phorbol-12-myristate-13-acetate (PMA), okadaic acid,1α,25-dihydroxyvitamin D3, 12-deoxyphorbol-13-acetate (prostratin),1,2-dioctanoyl-sn-glycerol (DOG), 1-oleoyl-2-acetyl-sn-glycerol (OAG),(2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam(α-amyloid precursor protein modulator), cis-9-octadecenoic acid (oleicacid), ingenol 3-angelate, resiniferatoxin,L-α-Phosphatidyl-D-myo-inositol-4,5-bisphosphate, triammonium salt(PIP2), phorbol-12,13-dibutyrate,8(S-hydroxy-(5Z,9E,11Z,14Z)-eicosatetraenoic acid (8(S)-HETE),12β-[(E,E)-5-Phenyl-2,4-pentadienoyloxy]daphnetoxin (merzerein),clomiphene citrate, sodium oleate, phorbol 12,13-diacetate,phorbol-12,13-didecanoate, 1,2-dipalmitoyl-sn-glycerol,1-Stearoyl-2-linoleoyl-sn-glycerol, phorbol-12,13-didecanoate,1,2-dipalmitoyl-sn-glycerol, 1-stearoyl-2-linoleoyl-sn-glycerol, phorbol12,13-dihexanoate, prostratin and its analogs, resiniferonol9,13,14-ortho-phenylacetate, C-8 ceramide,1,6-bis(Cyclohexyloximinocarbonylamino)hexane;1,6-Di(O-(carbamoyl)cyclohexanone oxime)hexane (RHC-80267),(+/−)-1-oleoyl-2-acetylglycerol,5(S),6(R),15(S)-TriHETE (Lipoxin A4),(−)-Indolactam V, SC-9, SC-10, zoledronic acid monohydrate,12-deoxyphorbo 13 -angelate 20-acetate,6-(N-decylamino)-4-hydroxymethylindole, 4α-phorbol 12,13-dibutyrate,1,2-dihexanoyl-sn-glycerol, zoledronic acid disodium salt tetrahydrate,arachidonic acid methyl ester, arachidonic acid-d8. In some embodiments,any of the above bryologs, potassium channel activators, or neristatinsmay be excluded from the method. Alternatively, in some embodiments, anyof the above bryologs, potassium channel activators, or neristatins maybe used in combination with one or more bryostatins in the methoddescribed above.

As used herein, “a pharmaceutically effective amount” is an amount of apharmaceutical compound or composition having a therapeutically relevanteffect on Alzheimer's disease. A therapeutically relevant effecttypically relates to or is evidenced by at least some improvement in abiomechanical process (e.g., gait, use of limbs, and the like) or achange in the cellular, physiological or biochemical parametersassociated with any of the causes of Alzheimer's disease. Atherapeutically relevant effect may also be evident in an improvedcognitive ability, such as short-term memory or problem solving.

The PKC activator is typically within a pharmaceutically acceptablecarrier (i.e., diluent) when administered. As used herein, and asgenerally understood in the pharmaceutical arts, the term“pharmaceutically acceptable” generally refers to those compounds,materials, compositions, and/or dosage forms that are, within the scopeof sound medical judgment, suitable for entering a living organism orliving biological tissue, preferably without significant toxicity,irritation, or allergic response. The pharmaceutical compositions of thepresent invention may be formulated for administration in liquid orsolid form. In the pharmaceutical composition, the compound is generallydispersed in the physiologically acceptable carrier, by being dissolvedor emulsified in a liquid carrier, or mixed (i.e., blended orcompounded) with a solid carrier. The carrier should be compatible withother ingredients of the formulation and physiologically safe to thesubject. Examples of suitable aqueous and non-aqueous carriers that maybe employed in the pharmaceutical compositions of the invention include,for example, water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), vegetable oils (such asolive oil), injectable organic esters (such as ethyl oleate), andsuitable mixtures thereof. In different embodiments, the pharmaceuticalformulation may be formulated for oral administration (e.g., as tablets,capsules, powders, granules, pastes, solutions, suspensions, drenches,or syrups); parenteral administration (e.g., by subcutaneous,intramuscular or intravenous injection as provided by, for example, asterile solution or suspension); sublingual or buccal administration;transdermal administration; or nasal administration. The pharmaceuticalcomposition may also include one or more pharmaceutically inactiveauxiliary agents, such as pH buffering agents; sugars (e.g., lactose,glucose, sucrose, and oligosaccharides, such as sucrose, trehalose,lactose, or dextran); antimicrobials; and/or sweetening, flavoring, orcoloring agents.

In certain embodiments, a pharmaceutically effective amount forbryostatins and/or bryologs may be from about 0.0000001 to about 5.00mcg per kg host body weight per day, which can be administered in singleor multiple doses. In some embodiments, the dosage level may be: fromabout 0.0000001 mcg/kg to about 2.50 mcg/kg per day; from about0.0000005 mcg/kg to about 1.00 mcg/kg per day; from at least about0.0000001 mcg/kg to about 2.50 mcg/kg per day; from at least about0.00000005 mcg/kg to about 1.00 mcg/kg per day; from at least about0.000001 mcg/kg to about 5.0 mcg/kg per day; or from about 0.00001mcg/kg to about 5.0 mcg/kg per dose. In other embodiments, the dosagemay be about 0.00000001 mcg/kg to about 0.00005 mcg/kg; 0.00005 mcg/kgto about 0.05 mcg/kg; about 0.0005 mcg/kg to about 5.0 mcg/kg per day;about 0.0001 mcg/kg to about 0.5 mcg/kg per dose; or 0.001 to 0.25mcg/kg per dose.

In some embodiments, a pharmaceutically effective amount of a PKCactivator may be an amount ranging from about 1 mcg to about 500 mcg perdose, or more particularly, from about 5 mcg to about 200 mcg per dose,or more particularly, from about 10 mcg to about 100 mcg per dose, ormore particularly, from about 20 mcg to about 40 mcg per dose. Indifferent embodiments, the PKC activator is administered in an amount ofprecisely or about 10, 15, 20, 25, 30, 35, 40, 45, or 50 mcg, or in anamount within a range bounded by any two of the foregoing values,wherein the term “about” generally indicates no more than ±10% or ±5%from a given value.

In some embodiments, the dosing is from about 1 μg/kg (3-25 μg/^(m2)) to120 μg/kg (360-3000 μg/m²). In other embodiments, the dosing is fromabout 0.04-0.3 μg/kg (1 μg/m²) to about 1-10 μg/kg (25 μg/m²). In otherembodiments, the dosing is from about 0.01 μg/m² to about 25 μg/m². Inother embodiments, the dosing is from about 0.0002-0.0004 μg/kg to about0.05-1 μg/kg.

In some embodiments, the PKC activator is a bryostatin or analoguethereof and a pharmaceutically effective amount of the bryostatin oranalogue thereof may be an amount ranging from about 1 mcg to about 500mcg per dose, or more particularly, from about 5 mcg to about 200 mcgper dose, or more particularly, from about 10 mcg to about 100 mcg perdose, or more particularly, from about 20 mcg to about 40 mcg per dose.In some embodiments, the PKC activator is a bryostatin or analoguethereof administered at a dosage of about 0.001 to 100 mcg/kg; 0.01 toabout 50 mcg/kg; about 0.1 to about 10 mcg/kg.

In some embodiments, the PKC activator is a bryostatin or bryolog, andthe bryostatin or bryolog is used (administered) in an amount from about0.0001 to about 1000 micrograms. In some embodiments, the bryostatin orbryolog is used in an amount of at least or about 0.0001, 0.0005, 0.001,0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0,150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, or1000.0 micrograms, or an amount within a range bounded by any two of theforegoing values.

In some embodiments, the PKC activator present in the compositions usedin the methods of the present disclosure is a bryostatin or bryolog, andthe bryostatin or bryolog is used (administered) in an amount of lessthat 50 micrograms (i.e., 50 mcg, or 50 μg). In some embodiments, thebryostatin or bryolog is used in an amount of less than 45 mcg. In someembodiments, the bryostatin or bryolog is used in an amount of less than40 mcg (40 μg). In other embodiments, the bryostatin or bryolog is usedin an amount of less than 30 mcg (μg). In further embodiments to any ofthe foregoing embodiments, a lower limit of at least or above 1, 2, 3,4, 5, 10, 15, 20, or 25 mcg may be used.

In some embodiments, the pharmaceutical composition will includebryostatin (e.g., bryostatin-1) in an amount of 0.1 mcg to 50 mcg, 0.1mcg to 25 mcg, 0.1 mcg to 20 mcg, 0.1 mcg to 15 mcg, 0.5 mcg to 50 mcg,0.5 mcg to 25 mcg, 0.5 mcg to 20 mcg, 0.5 to 15 mcg, 1 mcg to 50 mcg, 1mcg to 25 mcg, 1 mcg to 20 mcg, 1 mcg to 15 mcg, 1.5 mcg to 50 mcg, 1.5mcg to 25 mcg, 1.5 mcg to 20 mcg, 1.5 mcg to 15 mcg, 2 mcg to 50 mcg, 2mcg to 25 mcg, 2 mcg to 20 mcg, 2 mcg to 15 mcg, 2.5 mcg to 30 mcg, 2.5mcg to 25 mcg, 2.5 mcg to 20 mcg, 2.5 mcg to 15 mcg, 3 mcg to 25 mcg, 3mcg to 20 mcg, or 3 mcg to 15 mcg, typically within a pharmaceuticallyacceptable carrier. Moreover, any of the foregoing amounts may alsoserve as a dosage, which may be administered, for example, once or twiceper day, or by any other suitable regime as discussed above.

In some embodiments, the pharmaceutical composition include bryostatin(e.g., bryostatin-1) in an amount of 5 mcg to 20 mcg, 5 mcg to 10 mcg, 4mcg to 6 mcg, 6 mcg to 8 mcg, 8 mcg to 10 mcg, 10 mcg to 12 mcg, 12 mcgto 14 mcg, 14 mcg to 16 mcg, 16 mcg to 18 mcg, or 18 mcg to 20 mcg. Insome embodiments, the pharmaceutical compositions used in the methodsinclude bryostatin (e.g., bryostatin-1) in an amount of 0.1 mcg, 0.25mcg, 0.5 mcg, 1 mcg, 2.5 mcg, 3 mcg, 4 mcg, 5 mcg, 7 mcg, 7.5 mcg, 10mcg, 12.5 mcg, 15 mcg, 17.5 mcg, or 20 mcg. Moreover, any of theforegoing amounts may also serve as a dosage, which may be administered,for example, once or twice per day, or by any other suitable regime asdiscussed above.

The pharmaceutical compositions used in the methods of the presentdisclosure may be administered by any suitable regimen, such as aregimen of 1 to 4 times per day. In some embodiments, the compositionsare administered twice a week, once a week, once every two weeks, onceevery three weeks, once every four weeks, once every six weeks, onceevery eight weeks or less or more frequently depending on the needs ofthe patient. In some embodiments, the compositions used in the methodsof the present disclosure may be administered as part of a course oftreatment lasting for about 1 to about 30 days; about 1 to about 90days; about 1 to about 120 days; about 1 to about 180 days; about 1 to365 days; one year; two years; three years; or for the patient'slifetime. In some embodiments, the compositions used in the methods ofthe present disclosure may be administered as part of a course oftreatment lasting for at least about 5 weeks; at least about 9 weeks; atleast about 13 weeks; at least about 15 weeks. Nevertheless, thespecific dose level and frequency of dosage for any particular host maybe varied and will depend upon a variety of factors, including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the nature of the disorder, the severity of the particulardisorder, and the host undergoing therapy.

To facilitate a better understanding of the present invention, thefollowing examples of specific embodiments are given. In no way shouldthe following examples be read to limit or define the entire scope ofthis disclosure.

EXAMPLES Example 1

In a double-blind, randomized, placebo-controlled trial, bryostatin wasadministered by intravenous infusion (˜45 minutes) to patients withadvanced Alzheimer's disease for 12 weeks at doses of 20 or 40 μg.Randomization was stratified at baseline by severe (MMSE-2, 4-9) andmoderate (MMSE-2, 10-15) dementia, wherein MMSE-2 refers to Mini-MentalState Examination, version 2. The MMSE or Folstein test is a 30-pointquestionnaire that is used extensively in clinical and research settingsto measure cognitive impairment. Low scores indicate greater impairment.Version 2 of the MMSE was published in 2010, expanding the originalquestionnaire's usefulness in populations with milder forms of cognitiveimpairment. Safety results were similar to placebo for 20 μg with moreadverse events in the 40 μg group. The 20 μg dosing arm, but not the 40μg dosing arm, showed sustained efficacy with respect to baseline aswell as with respect to the Placebo group—as measured by positivechanges in the Severe Impairment Battery (SIB). Importantly, thiscognitive improvement persisted and increased 30 days after allbryostatin dosing had been completed. This improvement did not occur inthe presence of memantine (NMDA receptor antagonist) background therapy.

Methods

Adults aged 55-85 with cognitive deficits present for at least 2 years,MMSE-2 of 4-15, inclusive, and a diagnosis of Alzheimer's dementia wererandomly assigned to receive blinded intravenous infusions of bryostatin24 μg or matching placebo at weeks 0 and 1. The 24 μg and 48 μg dosesduring the first two weeks of the 20 and 40 μg protocols respectivelywere considered to be “loading” doses thought to be beneficial fromprior experience with Compassionate Use trial patients. Nelson et al.,Alzheimers Dis. 2017 58: 521-535. In one arm, the two 24 μg doses werefollowed by 20 μg doses at weeks 5, 7, 9 and 11 with the SevereImpairment Battery (SIB) measured at baseline and weeks 5, 9, 13, and15. In a second arm, the two 48 μg doses at weeks 0 and 1 were followedby 40 μg doses at weeks 5, 7, 9, and 11 with the SIB measured atbaseline and weeks 5, 9, 13, and 15. The subjects were stratified byMMSE-2 in two strata: Stratum 1 (4-9) and Stratum 2 (10-15). The firstprimary endpoint was safety and tolerability, assessed by treatmentemergent adverse events (TEAEs) with a sample size of 150 subjectsrandomized 1:1:1. Although efficacy was the secondary measure, theprimary efficacy endpoint was SIB improvement at week 13 evaluated usinga mixed model for repeated measures. A secondary efficacy endpoint wasADCS-ADL Sev. The primary safety population included all subjects whoreceived at least 1 dose of study drug, and efficacy was evaluated in amodified intent to treat (mITT) population that received at least 1 doseof study drug and had at least one treatment evaluation. Efficacy wasevaluated both as a primary end-point in the modified intent-to-treat(mITT) population and also as a primary end point in a Completerspopulation that included all patients that completed the entireprotocols as described above.

Results

Patients who received the 20 μg dose, but not patients receiving the 40μg dose, showed a sustained improvement in SIB versus baseline as wellas the placebo patients throughout the 13-week trial (FIG. 1). It ishypothesized that the lack of efficacy of the higher, 40 μg, dose wasdue to prolonged down regulation of PKC that typically follows higherand/or longer levels of PKC activation. Nelson et al., J Biol Chem.;2009 284(50):34514-21. As specified in the Statistical Plan that wasapproved by the FDA for this exploratory trial, all p-values are 1-sidedexcept as otherwise specified, as for example for correlation p-values(see below), post-hoc trend analyses, and post-hoc non-Namenda SIBimprovement analyses based on direct serial regressions, which have2-sided p-values. The results presented include Top Line, initialresults, analysis of exploratory endpoints, and analyses of post-hocendpoint results.

Sensitivity Analyses

Several sensitivity analyses were conducted and validated as describedbelow. MMRM (mixed model for repeat measures) analyses for Completerswere largely consistent with the corresponding FAS analyses acrossseveral key endpoints, including change-from-baseline scores for SIBTotal, ADCS-ADL SEV Total, and NPI. MMRM analyses for MMSE-2 and CGI-Igenerally showed less separation among treatment groups than wasobserved for the corresponding MMRM analyses for SIB Total Score.Analysis of Covariance (ANCOVA) and MMRM models for SIB Total Score andADCS- ADL Total Score were generally similar across various sensitivityanalyses. Standard errors for the Treatment Difference across endpoints(Changes Scores for SIB Total, ADCS-ADL Total, MMSE-2, and NPI) andModels (ANCOVA/MMRM with and without Site, including efficacy ofacetyl-cholinesterase-inhibitor (AChEI) as a factor, and including onlylarger sites) were very similar compared to the corresponding standarderrors for the treatment differences in the main models.

Several additional sensitivity analyses included a random site term gaveresults which were very similar to the results for the fixed effectsmodel. This reinforces reliance on the primary model, which reasonablyaccounts for the effect of dropouts, the impact of sites, and baselinecovariates. Using a quantitative baseline MMSE variable resulted in aslight improvement in sensitivity, but the results were very similar tothe results using a categorical factor for baseline MMSE stratification.A sensitivity analysis using z-score carried forward (using twoiterations) to account for dropouts and increase power was alsoconducted. The first iteration of the z-score imputation shows someseparation of the treatment groups as compared to the original meansfrom the MMRM model. The second iteration of the z-score imputationshowed results which were very similar to the results from the firstiteration, indicating that two iterations was sufficient. Although therewas some improvement in power, the results were similar enough to theresults from MMRM that the use of z-score imputation was not needed.Correction for baseline NPI Total Score was done in the primary mixedmodel to assess the possible impact. Forest plots show better effectsfor some items, especially for the 40 μg dose. The results acrossendpoints were much more consistent with each other than the resultswithout the correction for baseline NPI.

Significant SIB Improvement at 5 Weeks was Correlated with Weeks 9 and13 Efficacy

By week 5 (after 3 bryostatin doses had been administered), patients inthe 20 μg arm showed a significant improvement (FIG. 1) in the SIBscores compared to the placebo patients and compared to their baselinescores (p=0.016, Completers' Group; p=0.056; FAS).

As shown below (Table 1), the magnitude of the change scores in the 20μg group at 5 weeks was significantly correlated with the change scoresobserved at 9 and 13 weeks (p<0.001). These and other between-weekcorrelations support the consistency of the SIB improvements produced bythe 20 μg doses throughout the trial. These correlations also suggestthat the patients who showed improvement at 5 weeks (p=0.016) were thesame patients who showed improvement at 9 and 13 weeks. The placebogroup showed similar between visit correlations with the strongestcorrelations between weeks 5, 9, and 13 (p<0.001).

The correlations between visits were generally not as strong in the 40μg group (found to produce no SIB improvement) at any of the visits. Thecorrelations and p-values for the change scores for SIB (and ADC—poorbetween-group correlations) for the 20 μg group are given in Table 1below.

TABLE 1 Correlations and p-values for Total SIB and ADCS-ADL ChangeScores by Visit for the Bryostatin 20 μg group Table 1: Bryostatin SIBSIB SIB ADL ADL ADL 20 μg Week 5 Week 9 Week 13 Week 5 Week 9 Week 13SIB Week 5 Correlation 1 0.54375 0.52677 0.24549 0.11371 0.05365 p-value0.0004 0.0007 0.1126 0.5028 0.7525 SIB Week 9 Correlation 1 0.770130.28524 0.41538 0.34885 p-value <.0001 0.087 0.0106 0.037 SIB Week 13Correlation 1 0.24172 0.2795 0.15441 p-value 0.1495 0.0988 0.3615 ADLWeek 5 Correlation 1 0.75136 0.58849 p-value <.0001 0.0001 ADL Week 9Correlation 1 0.70214 p-value <.0001 ADL Week 13 Correlation 1

At 30 Days Post-Dosing (Week 15), the 20 μg Group Showed SIBImprovement:

The SIB efficacy measurement at 30 days (week 15) post dosing—after alldosing had been completed (last dose administered at week 11)—was apre-specified exploratory endpoint. Week 15 results were only includedfor subjects who were not re-randomized. The mixed model assumes missingat random, but order of enrollment in the trial was not random. Sinceorder of enrollment was not random, the assumption of missing at randomis not met. For this reason, only including week 15 for those who werenot re-randomized is an appropriate way to estimate week 15 results, butnot an appropriate way to estimate results at the earlier visits. Forthis reason, estimates up through week 13 are from a mixed model thatonly included data through week 13 and the week 15 estimates are from aseparate model. Results were generated using a mixed model with repeatedmeasures predicting change from baseline in SIB Total Score as theresponse variable and using baseline score, baseline MMSE strata,treatment, visit (categorical), and visit (treatment) as predictors.

The 20 μg patients (including week 15 for all subjects who were notre-randomized—FAS) showed a consistent improvement at week 15(approximately 30 days after the last scheduled dose of bryostatin at 11weeks) in their SIB scores over baseline and as compared to the Placebopatients (p =0.0503). In contrast to the 15-week SIB LS Mean improvementof 1.77, the Placebo patients showed a decline in their LS Mean SIBscores of 1.82 (with a treatment difference of 3.59) points fromBaseline to week 15 (see Table 2 and FIG. 2). For the Completers group,there was a persistent improvement (30 days after the last scheduleddose of bryostatin at 11 weeks) in their SIB scores over baseline and ascompared to the Placebo patients (p=0.0293). This corresponds to atreatment difference of the 20 μg patients from Placebo patients at 15weeks of 4.09 (See FIGS. 2 and 3).

TABLE 2 By Visit SIB Total: MMRM for Bryostatin 20 μg vs. PlaceboBryostatin Bryostatin Time Placebo - 20 μg - Placebo - 20 μg - Point FASFAS Completer Completer Week 5 n 46 44 42 38 LS Mean (SE) −1.77 (1.496)1.18 (1.102) −2.33 (1.583) 1.67 (0.964) Difference vs. 2.96 4.00 Placebop-value 0.0563 0.0164 80% CI for the 0.58, 5.34 1.63, 6.38 differenceWeek 9 n 43 38 42 37 LS Mean (SE) −0.09 (1.469) 0.94 (1.149) −0.57(1.539) 1.28 (1.119) Difference vs. 1.03 1.85 Placebo p-value 0.28980.1650 80% CI for the −1.36, 3.42  −0.59, 4.29  difference Week 13 n 4238 42 38 LS Mean (SE) −0.79 (1.328) 1.16 (1.150) −1.12 (1.387) 1.51(1.118) Difference vs. 1.94 2.63 Placebo p-value 0.1340 0.0699 80% CIfor the −0.31, 4.19  0.35, 4.91 difference Week 15* n 27 26 27 26 LSMean (SE) −1.82 (1.727) 1.77 (1.342) −2.13 (1.758) 1.96 (1.255)Difference vs. 3.59 4.09 Placebo p-value 0.0503 0.0293 80% CI for the0.79, 6.39 1.33, 6.85 difference *Results from Week 15 are from a modelthat included all visits.

Dose Efficacy is Supported When Efficacy is Referenced to Body SurfaceArea Analysis (BSA)

Normalization of the 20 μg doses to each patient's BSA (body surfacearea) revealed that the 20 μg doses—on a per-patient-basis were tightlydistributed around the 12.5 μg/m² (20 μg) dose, whereas the 40 μg doseson a per-patient-basis were broadly distributed around 25 μg/m². Theweek 13 mean dose adjusted for body surface area was 11.33 μg/m² in the20 μg dose group and was 21.34 μg/m² in the 40 μg dose group. Thesedifferences in distribution, as quantified by the F ratio (the ratio ofthe variance measures for 20 μg and the 40 μg dose distributions)suggest that the 20 μg dose patients may have received effective doselevels—at the frequency used in this protocol (cf. AAIC, 2017). Theratio of the variance for the 40 μg group divided by the variance forthe 20 μg group results in an F-ratio of 3.97 and a corresponding2-sided p-value of <0.0001, supporting a conclusion of unequalvariances. See FIG. 8. Comprehensive BSA results for both bryostatindose groups by visit showed that results for completers were similar tothose from the mITT population.

FIG. 8 shows the distribution with BSA normalization for both bryostatindose arms for subjects with SIB Total scores at Week 5. The figures foreach additional follow-up visit are similar since the BSA data isunchanged. The only difference between figures is that subjects who weremissing SIB Total score at the specified visit were not represented inthe figure for that visit.

Memantine Blocks Bryostatin SIB Improvement

Results were generated using a mixed model with repeated measurespredicting change from baseline in SIB Total Score as the responsevariable and using baseline score, baseline MMSE strata, treatment,visit (categorical), visit*treatment, memantine use*treatment, andmemantine use*treatment*visit as predictors. Again, results from week 15were obtained from a separate model that included week 15 data forsubjects who were not re-randomized. Results up through week 13 werefrom a model that included data up through week 13 for all subjects. Adifference in SIB efficacy was found in patients treated with 20 μgbryostatin with or without memantine (Namenda) as background SOCtherapy. The 20 μg patients (mITT) who were on memantine, a partial NMDAreceptor antagonist, showed no improvement in their SIB scoresthroughout the week 13 observation period when compared to placebopatients (20 μg LS Mean change=−0.60 versus −0.46 for placebo, p=0.4752for difference). In contrast, the 20 μg without memantine showedimprovement throughout the week 13 observation period that was greaterthan the SIB improvement previously reported (AAIC, 2017) for the entire20 μg group (see Table 3 and FIG. 4 below). The 13-week LS Mean changein the 20 μg group without memantine was 3.83 points compared to theplacebo group LS Mean change of −1.29, with a difference of 5.11 and ap-value of 0.0437. For the Completers patient group, the 13-week LS Meanchange in the 20 μg group without memantine showed no improvement intheir SIB scores throughout the 13 week observation period when comparedto placebo patients (20 μg LS Mean change =0.56 for placebo, p =0.3988for difference). In contrast, without memantine, the 13-week LS Meanchange in the 20 μg (Completers) was 4.22 compared to the Placebo groupLS Mean change of −1.32 with a difference of 5.53 and a p-value of0.0338.

The 15-week LS Mean change in the 20 μg group (mITT) without memantinewas 5.88 points compared to the placebo group LS Mean change of−0.05,with a difference of 5.93 and a p-value of 0.0576. This comparisonin the completer population had a difference of 6.36 and a p-value of0.0488. Results for the completer population, therefore, were generallysimilar to the FAS population.

TABLE 3 By Visit SIB Total: MMRM with Covariate Interactions IncludingMemantine Use FAS Completer Without Memantine With Memantine WithoutMemantine With Memantine Time Bryostatin Bryostatin BryostatinBryostatin Point Placebo 20 μg Placebo 20 μg Placebo 20 μg Placebo 20 μgOverall    14 (30.4%)   17 (38.6%)    32 (69.6%)    27 (61.4%)    13(31.0%)   16 (42.1%)    29 (69.0%)    22 (57.9%) Patient Counts, n (%)Week LS −1.74 (2.760) 2.74 (1.786) −1.70 (1.807)  0.26 (1.392) −2.16(2.898) 3.22 (1.454) −2.33 (1.925)  0.61 (1.224) 5 Mean (SE) Differ-4.48  1.96  5.38  2.94  ence vs. Placebo p-value 0.0857 0.1973 0.04870.1016 80% CI 0.29, 8.67 −1.00, 4.91 1.24, 9.51 −0.02, 5.91 for thediffer- ence Week LS  0.87 (2.714) 2.95 (1.804) −0.42 (1.773) −0.33(1.444)  0.69 (2.810) 3.34 (1.695) −1.06 (1.866) −0.16 (1.410) 9 Mean(SE) Differ- 2.08  0.09  2.66  0.90  ence vs. Placebo p-value 0.25970.4847 0.2071 0.3522 80% CI −2.08, 6.23  −2.87, 3.05 −1.53, 6.84  −2.14,3.93 for the differ- ence Week LS −1.29 (2.449) 3.83 (1.733) −0.46(1.608) −0.60 (1.425) −1.32 (2.546) 4.22 (1.639) −0.96 (1.688) −0.39(1.384) 13 Mean (SE) Differ- 5.11  −0.14   5.53  0.56  ence vs. Placebop-value 0.0437 0.4752 0.0338 0.3988 80% CI 1.30, 8.93 −2.92, 2.65 1.68,9.38 −2.27, 3.40 for the differ- ence Week LS −0.05 (3.151) 5.88 (2.036)−1.61 (2.304) −0.83 (1.703) −0.12 (3.225) 6.24 (2.046) −2.16 (2.367)−0.71 (1.728) 15* Mean (SE) Differ- 5.93  0.79  6.36  1.45  ence vs.Placebo p-value 0.0576 0.3927 0.0488 0.3120 80% CI  1.12, 10.73 −2.93,4.50  1.47, 11.25 −2.36, 5.25 for the differ- ence

As described above, overall, the 15-week LS Mean change for the 20 μggroup was 1.77 points, compared to the placebo group LS Mean change of-1.82 points with a difference of 3.59 (p=0.0503). Memantine partiallyblocks the NMDA glutamate post-synaptic receptor that is well-known tobe regulated by PKC phosphorylation. This regulation may explainprevention of bryostatin's therapeutic efficacy for the patients onmaintenance doses of Memantine throughout this trial.

Efficacy of Secondary Psychometric, ADCS-ADL

As for the SIB, the ADCS-ADL efficacy was significant for the 20 μg butnot the 40 μg dose (FIG. 6). For the μg dose, the mITT group showed adifference from placebo of 1.4 (p<0.104), while for the completers group(20 μg) showed a difference from placebo of 1.6 (p<0.082). Subgroupswith and without memantine (Namenda)—as background SOC therapy—were notsignificantly different above placebo.

There was no evidence of treatment by efficacy ofacetyl-cholinesterase-inhibitor (AChEI) use interaction. For SIB Total,the two sided p-value for the interaction between treatment and AChEIuse was 0.8244 and the two sided p-value for the interaction betweentreatment, AChEI use and time was 0.9223. Similar results were seen wheninvestigating the effect of AChEI use on treatment effect for ADCS-ADLwith p-values of 0.5137 and 0.6256 (two sided), respectively. At week13, the LS Mean difference between 20 μg group and placebo in SIB Totalfor AChEI non-users was 2.00 and users was 2.09, and in ADCS-ADL forAChEI non- users was −0.67 and users was 1.99.

Sustained SIB Benefit Reinforced by Combined Analyses

(A) MMRM-A combined treatment effect across Weeks 5, 9, and 13 wascalculated as a summary of the overall treatment effect. The estimatedcoefficients from the MMRM were used to compute and average threedeltas:

Combined=(ΔWeek 5+ΔWeek 9+ΔWeek 13)/3

The difference in the combined average treatment effects between 20 μgand placebo groups was estimated and tested for significance using LeastSquare Means. For the combined analyses at week 13, the bryostatin 20 μggroup in the FAS with the post-hoc MMRM analysis showed the following:

The combined treatment effect from Weeks 5, 9, and 13 showed a meanimprovement in SIB of 2.146 (1-sided p=0.093) for bryostatin 20 μg overplacebo in the FAS population, and a mean improvement of 3.089(1-sidedp=0.032) over placebo in the Completer population.

Using the post-hoc combined analysis method, results for both FAS andcompleter populations show numerically greater improvement of bryostatin20 μg over placebo, and reached statistical significance based on theprotocol pre-specified 1-sided α=0.10 level.

By showing the summary statistic for the entire treatment period, ratherthan at an arbitrary specific time point, the results offer a moreclinically relevant interpretation—particularly the sustained nature ofthe SIB improvement caused the cumulative treatment benefits ofbryostatin 20 μg.

An additional supplemental post-hoc analysis was conducted using thefollowing SAP-defined exploratory endpoint: Change from baseline in SIBScore at the 30-day follow-up (Week 15 visit). The post-hoc MMRManalysis was performed on the population of patients who have a 30-DayFollow-Up Visit (N=26 for bryostatin 20 μg, and N=27 for placebo for the17-Week Completer Set) Using the post-hoc analysis method on the 30-DayFollow-up population of patients yields the following:

The combined treatment effect from Weeks 5, 9, 13, and 15 shows a meanimprovement in SIB of 4.721 (1-sided p=0.032) for bryostatin 20 μg overplacebo. This magnitude of improvement in SIB change is substantiallylarger than the improvements seen in all previous analyses.

The estimated improvement of bryostatin 20 μg over placebo is greater ateach week and for the entire treatment period for this population, andreached statistical significance based on the protocol pre-specified1-sided α=0.10 level at each visit.

These supplemental analyses of the change in baseline SIB Score at the30-day follow-up exploratory endpoint further suggests that thetreatment benefits of bryostatin 20 μg persist after the treatmentperiod ends.

(B) Simple Comparisons—Post-hoc analyses of SIB scores, for the placeboand low dose bryostatin (20 μg dose) treatment arms, stratified byexposure to memantine.

A total of 57 patients with exposure to memantine (31 placebos and 26low dose bryostatin patients) and 33 memantine treatment-naive patients(15 placebos and 18 low dose bryostatin patients) were considered in thepost-hoc analyses. Patients used here are from the modified ITT FASsample (e.g. patients with at least one post-baseline SIB).

Unadjusted Analysis of the Primary Efficacy Endpoint: The primaryefficacy endpoint for each individual patient is the change in averageof the SIB scores obtained in the 13 to 15-week time window from thebaseline SIB score. If a patient is missing from the study at either the13 week or 15- week time point, then the SIB for this time window isgiven as the one SIB value obtained. Since the number of patient missingat both the 13 week and 15-week time point was low, no SIB values wereimputed. A total of 7 memantine patients and 3 memantine naïve patientsin the FAS sample were missing from the study at both week 13 and week15.

The group difference was statistically assessed by considering the meanprimary efficacy endpoint averaged over all patients with endpoint datain each treatment arm. The t-test for two independent samples, assumingunequal variance, was performed on the group difference of these means.In addition, the Wilcoxon rank sum test was also performed to determinethe robustness of the t-test.

Tables 4 and 5 below show the treatment arm means (SD) of the primaryefficacy endpoint, and the corresponding two-sided p-values from boththe test and Wilcoxon tests. Statistical comparisons between treatmentdifferences in the means of the difference in baseline SIB and SIB attimes 3, 9, and 13 weeks are given for comparison purposes.

TABLE 4 Simple Comparison of the Primary Efficacy Endpoint by T-test andWilcoxon Test for the Memantine Treatment-Naïve Patients. Placebo Lowdose (20) T-test Wilcoxon test SIB Mean SD Mean SD T P-val W P-val Deltaat Week 5 −1.200 10.262 3.444 5.752 1.560 0.134 88 0.091 Delta at Week 90.786 7.444 3.467 7.039 0.995 0.329 76 0.203 Delta at Week 13 −1.1436.893 4.500 7.014 2.219 0.035 66 0.058 Delta at Ave of −0.679 6.7075.406 5.432 2.706 0.012 0.016 Week 13 and 15

TABLE 5 Simple Comparison of the Primary Efficacy Endpoint by T-test andWilcoxon Test for the Memantine Exposed Patients. Placebo Low dose (20)T-test Wilcoxon test SIB Mean SD Mean SD T P-val W P-val Delta at Week 5−1.258 10.498 0.077 8.109 0.541 0.590 385 0.779 Delta at Week 9 −0.41411.008 −0.087 6.522 0.133 0.895 328 0.919 Delta at Week 13 −0.464 10.031−0.545 6.508 −0.035 0.973 318 0.852 Delta at Ave of −1.768 10.306 −0.7276.388 0.438 0.664 1.000 Week 13 and 15

Adjusted Analysis of the Primary Efficacy Endpoint: Because the post-hocanalyses presented here focuses on the subgroup of patients defined bymemantine exposure, there can be no assumption that randomizationcontrolled for the potential imbalance of baseline factors between theplacebo and bryostatin treatment arms within the memantine exposurestrata. Thus, any possible difference between treatment arms within thememantine strata could be confounded by memantine exposure. For example,it is possible that bryostatin-treated patients in the memantinetreatment-naive group are more likely to be in the higher MMSE-2 strataversus memantine treatment-naive placebo, which in turn might result ina higher SIB endpoint for these patients.

Analyses of Covariance models were created to control for potentialbaseline imbalances. These models compare differences in the group meansof the primary efficacy endpoints (SIB at 13/15 weeks from baseline)with the memantine strata, while controlling for the SIB at baseline andthe MMSE-2 strata at randomization. Table 6 shows the regression resultsfor both memantine naïve and memantine exposed groups.

TABLE 6 Baseline SIB and MMSE-adjusted Slopes for Placebos and 20 μgBryostatin Patients by Memantine exposure. Memantine Naïve MemantineExposed Base SIB, Base SIB, Baseline SIB MMSE- 2 Baseline SIB MMSE- 2Treatment  5.426 (p = 0.010) 5.923 (p = 0.002) −0.531 (p = 0.843) −0.620(p = 0.815) (95% CI) (1.402, 9.451) (2.328, 9.518) (−5.877, 4.816)(−5.925, 4.685)  Baseline SIB −0.180 (p = 0.005) −0.243 (p < 0.001)  0.131 (p = 0.116) 0.048 (0.636)  (95% CI) (−0.302, −0.058) (−0.361,−0.126) (−0.034, 0.295) (−0.155, 0.252)  MMSE-2 6.790 (p = 0.008)  4.266(p = 0.183) (95% CI)  (1.942, 11.638) (−2.081, 10.613)

Analysis of Linear Trends in SIB over Time: In addition to consideringthe difference in treatment means of the change of SIB at each timepoint from the baseline, the change in SIB values over all the timepoints was also considered. Initially, the slope of each person'strajectory over all time points was estimated, from baseline (week 0) tothe week 15. The mean slopes were then calculated for treatment armwithin both memantine strata. The means of the treatment-specific slopeswere statistically compared using the two-sample t-test and Wilcoxonrank sum test.

Table 7 shows the mean (95% CI) of the slope for each treatment andmemantine exposure combination for the unweighted data as well as thesedata weighted by the number of non-zero SIB measures.

TABLE 7 Mean (95% CI) Slopes, both Unweighted and Weighted by the Numberof Non-Zero SIB scores. 20 μg t-test 20 μg t-test Placebo BryostatinWilcoxon Placebo Bryostatin Wilcoxon Unweight 0.152 0.382 P = 0.210−0.002 −0.143 P = 0.627 Slope (−0.124, 0.428) (0.130, 0.634) P = 0.047(−0.391, 0.387) (−0.568, 0.282) P = 0.911 Weighted 0.067 0.363 P = 0.067−0.050 −0.099 P = 0.831 Slope (−0.166, 0.300) (0.146, 0.581) (−0.355,0.254) (−0.437, 0.238)

Discussion:

The effects described above suggest that the 20 treatment-dose ofbryostatin can safely produce sustained improvements in SIB scores ofmoderate to severe AD patients based on SIB improvements measured atweek 13, and that these improvements may be sustained for weeks (e.g.,week 15) after the termination of the dosing protocol (week 11).Bryostatin produces enhanced SIB improvement in the absence ofmemantine, as shown in FIGS. 1-7 and Table 3 above. Patients onmemantine showed no SIB improvement at week 13 or 15 (See FIGS. 4, 5).Collectively, the overall weight and consistency of the data provide abasis for a cognitive-improvement efficacy of bryostatin for theadvanced AD patient.

These results, of an exploratory trial of bryostatin in the treatment ofadvanced Alzheimer's disease patients, are worth considering in thecontext of all prior drug trials for the treatment of advancedAlzheimer's patients.

Previous trials, ranging from neurotransmitter agonists and/orantagonists to anti-amyloid antibodies and to gamma secretaseinhibitors, have rarely if ever produced sustained benefit (vs. placebo)in advanced AD patients.

Here, in every analysis that was conducted, unadjusted, adjusted withMMRM, for 13 weeks and for 15 weeks (30 days post completion of all drugdosing) showed sustained improvement of the SIB vs. baseline and placebogroup patients. At 13 weeks for the Completers, the delta improvementwas 2.6; at 15 weeks, the improvement delta was 4.0; and at 15 weeks—inthe absence of memantine—the delta was 6.36 (See FIG. 7). According tothe pre-specified statistical plan, all of these delta values weresignificant.

In contrast to this observed improvement of SIB, most, if not all,trials directed toward disease modification, have had a differentimprovement objective: a reduction in the rate of decline of SIB (vs. ashift of decline to improvement). None of these trials has yetsucceeded.

The sustained improvement demonstrated with bryostatin began at 5 weeksin the Completers' group (p<0.016) that was correlated with highstatistical significance (p<0.001) with improvement at 9, 13, and 15weeks. These correlations suggest that the same patients benefitedthroughout the 15-week trial.

It is also worth emphasizing that the memantine blocked all benefit ofbryostatin in the SIB improvement. The principle targets of bryostatin,PKC isozymes, are known to regulate NMDA receptor functions (blocked bymemantine). Therefore, blockade of the NMDA receptor could offset mostif not all of the bryostatin-induced SIB improvement. PKC regulation ofthe NMDA receptor functions includes increasing NMDA conductance byrelieving Mg++ blockade, controlling trafficking of the NMDA receptor tothe neuronal membranes, and enhancing NMDA-induced synaptogenesis. Thissynaptogenesis, a primary mechanism of action (MOA) of bryostatindemonstrated in a variety of pre-clinical models, is mediated bybryostatin-PKC epsilon enhancement of several synaptic growth factorsthat include BDNF, NGF, and IGF.

The apparent bryostatin-induced persistence of SIB improvement isconsistent with a long-lasting consequence of PKC epsilon-growth factorefficacy that could induce the growth and/or maturation of synapticnetworks in the brain. The present example describes the first multiplebryostatin dose treatment of AD patients in a double-blind, randomized,placebo-controlled phase 2 trial for 12 weeks. The results describedherein surprisingly and unexpectedly suggest that a dose level of 20 mcgcan safely produce sustained improvements in the Severe ImpairmentBattery (SIB) scores of moderate to severe AD patients measured at week13. Moreover, these improvements may be sustained up to 4 weeks afterthe termination of the dosing protocol at 11 weeks. This efficacy wasonly apparent in the absence of baseline, standard of care memantinetherapy.

Example 2

The following prophetic example provides a further assessment of thesafety, tolerability and efficacy of bryostatin in the treatment ofmoderately severe to severe Alzheimer's disease subjects not receivingmemantine treatment. This randomized double-blind placebo-controlled,confirmatory study will compare bryostatin to placebo for the treatmentof moderately severe to severe Alzheimer's disease in subjects notreceiving memantine treatment. The study may be 15 weeks in duration,including a safety and efficacy evaluation 30 days after the last doseof study drug. Subjects will receive 7 doses of drug during the study.The primary efficacy endpoint will be the Severe Impairment Battery(SIB) scale score after 12 weeks of treatment (e.g., taking an averageof SIB measures observed during the Week 13 to Week 15 time window).Bryostatin-1 or matching Placebo will be administered intravenous bycontinuous infusion over 45±5 minutes.

Eligible subjects will be stratified based on Mini Mental State Exam(MMSE-2) scores 4-9 vs. 10-15 and will be randomized 1:1 to one of twotreatment arms: 20 μg bryostatin or placebo for twelve weeks. The firsttwo doses of study drug will be a loading dose 20% higher (i.e., 24 μg)than the assigned dose and will be administered one week apart.Thereafter, the assigned dose of 20 μg will commence with the third doseand be administered every other week. Drug is administered IV bycontinuous infusion over 45(±5) minutes. Subjects are scheduled toreceive seven doses over 12 weeks. Subjects who drop out prior tocompleting the Week 7 visit will be replaced, up to a maximum of 15subjects. Safety and tolerability may be determined through evaluationsof adverse events (AE), serious adverse events (SAE), physicalexamination (PE), vital signs, 12-lead electrocardiogram (ECG), theColumbia Suicide Severity Rating Scale (C-SSRS), and clinical laboratoryassessments.

Cognitive improvements will be evaluated primarily with the SevereImpairment Battery (SIB) and secondarily with functional improvements inthe Alzheimer's Disease Cooperative Study—Activities of Daily Living -Severe Impairment Version (ADCL-ADL-SIV), Clinical Global Impression ofImprovement (CGI-I), and Neuropsychiatric Inventory (NPI) metrics. Theprimary efficacy endpoint is defined as the SIB scale score obtainedbetween 13 and 15 weeks post first dose. The average SIB score at 13 and15 weeks will be the primary efficacy end point for patients with SIBoutcome measures at both time points; otherwise, either the 13-week or15-week SIB will be considered as the primary efficacy endpoint forpatients present in the study at only one of these two times. Theprimary efficacy analysis is based on the two-sample t-statistics of thetreatment group averages from the primary endpoint. A Wilcoxon test willalso be performed.

Patients treated with bryostatin may experience a greater improvement incognitive function as measured by the SIB from baseline to the primaryefficacy endpoint after 12 weeks of treatment, as compared to patientson placebo during the same time period. The null hypothesis will berejected at a significance level of a two-sided α=0.05. The test of thenull hypothesis will be a superiority test based on the two-samplet-test, and only an improvement in the SIB score from baseline is ofclinical significance.

One of skill in the art will appreciate that the examples herein are notintended to be limiting and that one of skill in the art will readily beable to apply the teachings herein to treating Alzheimer's disease.Therefore, the present methods and compositions are well adapted toattain the ends and advantages mentioned as well as those that areinherent therein. While numerous changes may be made by those skilled inthe art, such changes are encompassed within the spirit of thisdisclosure as illustrated, in part, by the appended claims.

1.-14. (canceled)
 15. A method for inducing synaptogenesis in a subjecthaving synaptic loss, the method comprising orally administering to thesubject a therapeutically effective amount of a synaptic growth factoractivating compound that functions as a PKC activator to result in anincrease in synaptogenesis in said subject, wherein the synaptic growthfactor activating compound is administered for the first two doses atabout 24 micrograms followed by four consecutive doses of about 20micrograms each.
 16. The method of claim 15, wherein the synaptic growthfactor is selected from the group consisting of brain-derivedneurotrophic factor (BDNF), nerve growth factor (NGF), and insulin-likegrowth factor (IGF).
 17. The method of claim 15, wherein the methodincreases BDNF levels.
 18. The method of claim 15, wherein the methodprevents cognitive loss.
 19. The method of claim 15, wherein the methodrestores lost synapses.
 20. The method of claim 15, wherein the synapticgrowth factor activating compound is combined with a non-growth factorPKC activator, wherein PKC is protein kinase C.
 21. The method of claim15, wherein the subject is not administered an N-methyl-D-aspartate(NMDA) receptor antagonist.
 22. The method of claim 15, wherein saidfour consecutive doses are administered every other week.
 23. The methodof claim 15, wherein said first two doses at about 24 micrograms arefollowed by at least four consecutive doses of about 20 micrograms each.24. A method for inducing synaptogenesis in a subject having synapticloss, the method comprising administering to the subject atherapeutically effective amount of a synaptic growth factor activatingcompound that functions as a PKC activator to result in an increase insynaptogenesis in said subject, wherein the synaptic growth factoractivating compound is administered intravenously as a continuousinfusion.
 25. The method of claim 24, wherein the synaptic growth factoractivating compound is administered intravenously as a continuousinfusion every other week.
 26. The method of claim 24, wherein thesynaptic growth factor activating compound is administered intravenouslyover 40-50 minutes.
 27. The method of claim 24, wherein the synapticgrowth factor activating compound is administered intravenously over40-50 minutes every other week.
 28. The method of claim 24, wherein thegrowth factor is selected from the group consisting of brain-derivedneurotrophic factor (BDNF), nerve growth factor (NGF), and insulin-likegrowth factor (IGF).
 29. The method of claim 24, wherein the methodincreases BDNF levels.
 30. The method of claim 24, wherein the methodprevents cognitive loss.
 31. The method of claim 24, wherein the methodrestores lost synapses.
 32. The method of claim 24, wherein the synapticgrowth factor activating compound is combined with a non-growth factorPKC activator, wherein PKC is protein kinase C.
 33. The method of claim24, wherein the subject is administered about 10 to about 100 μg of thesynaptic growth factor activating compound.
 34. The method of claim 24,wherein the subject is not administered an N-methyl-D-aspartate (NMDA)receptor antagonist.